CD43, a prevalent white blood cell molecule distinguished by its mucin- like surface region, has been proposed as a “functional barrier” that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3′4′- dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.